E. coli OmpT protease (SEQ ID NO: 41) is present in E. coli outer membrane fractions, and this protease selectively cleaves primarily peptide bonds between basic amino acid pairs. Proteins having homologous amino acid sequences with E. coli OmpT protease and having or believed to have protease activity are also found in intestinal bacteria such as Salmonella, Yersinia and Shigella, and this group of proteins is known as the omptin family.
E. coli OmpT protease (SEQ ID NO: 41) has a molecular weight of approximately 33,500. Sugimura et al. have examined the substrate specificity of OmpT protease (SEQ ID NO: 41) and have reported that the enzyme specifically cleaves the central peptide bonds between the basic amino acid pairs of arginine-arginine, lysine-lysine, arginine-lysine and lysine-arginine (Sugimura, K. and Nishihara, T. J. Bacteriol. 170: 5625-5632, 1988).
However, the enzyme does not cleave all basic amino acid pairs, as it is highly specific. For example, human γ-interferon contains 10 basic amino acid pairs, but only two of them are cleaved (Sugimura, K. and Higashi, N.J. Bacteriol. 170: 3650-3654, 1988). This is attributed to the influence of the three-dimensional structure of the human γ-interferon substrate and to the amino acid sequences of sites thought to be recognized by the enzyme which are adjacent to basic amino acid pairs.
The amino acid positions of substrates referred to throughout the present specification are assigned according to the notation method of Schechter and Berger (Schechter, I. and Berger, A. Biochem. Biophys. Research. Commun. 27: 157-162, 1967). That is, the peptide bond between the P1 position and P1′ position of Pn . . . P2-P1-P1′-P2′ . . . Pn′ is the cleavage site, and the amino acids are represented by their standard single letter or three-letter abbreviations, with ↓ indicating the cleavage site.
For example, if cleavage is between lysine and arginine of the amino acid sequence -leucine-tyrosine-lysine-arginine-histidine- (-Leu-Tyr-Lys↓Arg-His-) (SEQ ID NO: 9), leucine is at the P3 position, tyrosine is at the P2 position, lysine is at the P1 position, arginine is at the P1′ position, and histidine is at the P2′ position.
Also, unless otherwise specified, these designations will be used as the amino acid positions corresponding to the original sequence even when an amino acid substitution has been introduced at the cleavage site or its surrounding amino acid sequence such that it is no longer cleavable, or a new cleavage site has resulted.
OmpT protease cleavage sites have been discovered with amino acid sequences other than basic amino acid pairs, and Dekker et al., using substrates with amino acid substitutions introduced into an OmpT protease substrate comprising the amino acid sequence Ala-Arg-Arg-Ala (SEQ ID NO: 10) (P2-P1↓,P1′-P2′), have reported that OmpT protease exhibits high specificity for the basic amino acids arginine and lysine as the amino acid at the P1 position of the cleavage site, but is less stringent in regard to the amino acid at the P1′ position (Dekker, N. et al. Biochemistry 40: 1694-1701, 2001).
Moreover, the present inventors, using as the substrate a fusion protein capable of being cleaved by the enzyme under polypeptide-denaturing conditions in the presence of urea wherein an amino acid substitution is introduced at the P1′ position of the fusion protein, have discovered that cleavage occurs when the P1′ position amino acid is an amino acid other than aspartic acid, glutamic acid or proline (Okuno, K. et al. Biosci, Biotechnol. Biochem. 66: 127-134, 2002, Japanese Patent Application No. 2000-602803). Yet the cleavage efficiency in these cases is still lower than when the amino acid residue at the P1′ position is arginine or lysine.
As for the specificity with respect to the sequences adjacent to the cleavage site, it has been demonstrated that cleavage fails to occur when an acidic amino acid is present at the P2 or P2′ position (Dekker, N. et al. Biochemistry 40: 1694-1701, 2001).
The present inventors have also reported that cleavage efficiency is increased when arginine or lysine is present as a basic amino acid at the P4 position or P6 position, while conversely it is decreased in the case of an acidic amino acid such as aspartic acid or glutamic acid (Okuno, K. et al. Biotechnol. Appl. Biochem. 36: 77-84, 2002, Japanese Patent Application No. 2000-602803).
While the specificity for other sequences adjacent to the cleavage site has not been established, the fact that OmpT protease cleaves protamines, which are highly basic antimicrobial peptides (Stumpe, S. et al. J. Bacteriol. 180: 4002-4006, 1998), and that many acidic amino acids are found in the OmpT protease extracellular domain involved in protease activity (Vandeputte-Rutten, L. et al. EMBO J. 20: 5033-5039, 2001), suggests that charge effects are important for the interaction between OmpT protease and its substrate.
As regards applications of OmpT protease, the high cleavage site specificity and the fact that the protease is present on the outer membrane of E. coli means that the protease can be used as a processing enzyme for releasing target polypeptides from fusion proteins created by gene recombination techniques.
Hanke et al., in carrying out secretion of cholesterol esterase using E. coli, fused it with E. coli hemolysin A protein, secreted the fusion protein extracellularly and allowed OmpT protease on the outer membrane to act thereon, thereby successfully obtaining active cholesterol esterase from the fusion protein. Here, a linker with an arginine-lysine sequence was added for cleavage of the sequence with OmpT protease (Hanke, C. et al. Mol. General. Genet. 233: 42-48, 1992).
The present inventors have discovered that OmpT protease is resistant to denaturing agents, and have utilized this property to show that fusion proteins expressed as inclusion bodies can be cleaved in the presence of denaturing agents. Specifically, a Staphylococcus aureus V8 protease derivative fusion protein was expressed as an inclusion body in an E. coli expression system, solubilized with urea and then acted upon by OmpT protease in the presence of urea, which resulted in release of the V8 protease derivative portion from the fusion protein, and subsequent refolding allowed successful production of the V8 protease derivative with enzyme activity (Yabuta, M. et al. Appl. Microbiol. Biotechnol. 44: 118-125, 1995).
Normally, release of a target polypeptide or protein from a fusion protein is accomplished using an enzyme with high amino acid sequence specificity as the processing enzyme. The known proteases used in such cases include factor Xa, thrombin and enterokinase, but because these enzymes are mammalian derived enzymes and therefore in short supply and costly, they are not suitable for industrial mass processing of peptides and proteins by fusion protein methods. In addition, when the target polypeptide or protein is to be used as a pharmaceutical, it is also necessary to consider viral contamination originating from the enzyme source, as well as contamination by altered prion proteins which are causative factors of bovine spongiform encephalopathy.
Since OmpT protease is derived from E. coli, its use as a processing enzyme is clearly preferred over the aforementioned enzymes in terms of supply volume, cost and safety. Moreover, because OmpT protease is also present in inclusion bodies, it can act simply upon lysing the fusion protein with a denaturing agent such as urea even when the fusion protein is expressed as an inclusion body. Furthermore, OmpT protease is also present on the E. coli outer membrane and therefore OmpT protease reaction can be carried out by addition of the cells themselves to the reaction system (Grodberg, J. and Dunn, J. J. J. Bacteriol. 170: 1245-1253, 1988).
Most proteases used for processing of E. coli-produced fusion proteins to obtain target polypeptides, in industrial peptide production such as production of pharmaceuticals, are not derived from E. coli and must therefore be purified for use. A major improvement in polypeptide production cost could thus be afforded if OmpT protease could be used as the processing protease by mere addition of the outer membrane fraction or inclusion body lysis from E. coli cells themselves, without requiring purification. However, processing of fusion proteins using conventional E. coli OmpT protease, with the exception of a few cases, has been restrictive in that only polypeptides whose N-terminal amino acids are lysine or arginine are released.
Despite the usefulness of OmpT protease, knowledge has been limited, prior to the present invention, for the use of OmpT protease as a cleavage enzyme for fusion proteins, as regards how the sequence of the cleavage site and its adjacent amino acids should be designed in order to achieve specific and efficient cleavage at the intended site. Consequently, the types of N-terminal amino acids for efficiently cleavable target polypeptides have been limited. This has been a cause of problems including restrictions on the types of target polypeptides that can be obtained and resulting in, for example, the inability to accomplish efficient cleavage even when cleavage is possible.
Patent document 1: Japanese Patent Application No. 2000-602803
Non-patent document 1: Sugimura, K. and Nishihara, T. J. Bacteriol. 170: 5625-5632, 1988
Non-patent document 2: Sugimura, K. and Higashi, N.J. Bacteriol. 170: 3650-3654, 1988
Non-patent document 3: Schechter, I. and Berger, A. Biochem. Biophys. Research. Commun. 27: 157-162, 1967
Non-patent document 4: Dekker, N. et al. Biochemistry 40: 1694-1701, 2001
Non-patent document 5: Okuno, K. et al. Biosci, Biotechnol. Biochem. 66: 127-134, 2002
Non-patent document 6: Okuno, K. et al. Biotechnol. Appl. Biochem. 36: 77-84, 2002
Non-patent document 7: Stumpe, S. et al. J. Bacteriol. 180: 4002-4006, 1998
Non-patent document 8: Vandeputte-Rutten, L. et al. EMBO J. 20: 5033-5039, 2001
Non-patent document 9: Hanke, C. et al. Mol. General. Genet. 233: 42-48, 1992
Non-patent document 10: Yabuta, M. et al. Appl. Microbiol. Biotechnol. 44: 118-125, 1995
Non-patent document 11: Grodberg, J. and Dunn, J. J. J. Bacteriol. 170: 1245-1253, 1988